Optimization the enzymatic degradation of waste feather for FPL production and feather meal formation using YNDH protease/keratinase crude enzyme.
In order to achieve the optimum response area for maximizing the protein contents of enzymatically degraded feather in form of concentration (µg ml-1), the significant independent variables [X1; time (h), X2; feather concentration (g), X3; keratinase activity (U/ml)] were further explored, each at three levels. Table 1 represents the design matrix of the variables in coded units with the experimental results of the protein lysate concentration. All cultures were conducted in triplicates and the observations averaged.
Multiple regression analysis and ANOVA test
The three variables with fourteen trails were analyzed using linear multiple regression analysis approach and the percentage confidence rates (percent) were determined. The p-value from the ANOVA analysis for each response was determined to analyze the relationship between the variables at 90% or higher confidence level and the analysis of variance using ANOVA test in Box- Behnken experiment was generated and summarized in Table 2 which gives p=0.0235. Since the p-value indicated in the ANOVA Table is less than 0.05, it is concluded that there is a statistically significant relationship among the studied variables at 95% confidence level (p=0.05). The value of the determination coefficient R2 =0.953 for protein contents (concentration) of enzymatic feather-lysate, being a measure of fit of the model, indicates that about 4.7% of the total variations are not explained by protein contents concentration. The adjusted R-squared statistic is 0.85. Presenting experimental results in the form of surface plots (Fig. 1) show that higher levels of protein contents concentration were attained with the coded values 0.52 of time, 1 of substrate concentration and 0.457 of enzyme concentration. The optimum values of the three variables analyzed as obtained from the maximum polynomial model point were calculated using the JMP programme and found to be: time, 20.2h; substrate concentration, 3g/100ml and enzyme concentration, 24.57U/100ml with prediction calculated protein contents concentration equal to 2159.85 µg /ml. Bench scale experiments showed that Y value of 2089.5 µg ml-1 was obtained, this means the calculated model accuracy was 96.7 %. In this study a statistical technique, of Box–Behnken design was shown to be efficient and reliable in selecting the statistically significant factors and finding the optimal concentrations of those factors. Thefore, the following conditions are considered to be near the maximum, based on the results obtained from the Box-Behnken method: time 20.2h, substrate concentration, 3g%; enzyme concentration, 24.57U%; pH 10 and cultivation temperature 50oC, the protein contents concentration measured was 2159.85 µg/ml. Finally we can conclude that, protein contents of enzymatically degraded feather by effect of keratinase from Laceyella sacchari strain (YNDH) has been systematically improved during various experimental designs compared with basal medium.
Amino acid analysis of feather meal
In this experiment the feather lysate were analyzed for free amino acids released during feather degradation in both cell free supernatant after cultivation of laceyella sacchrii YNDH in optimized medium and feather hydrolysate produced due to enzymatic treatment as shown in Table 3 (A and B), respectively. 16 amino acids were detected in both experiments and quantified as shown in Table 3. Presence of essential amino acids like leucine and isoleucine indicates that feather treatment with the native isolate YNDH and YNDH crude keratinase enzyme provided feather lysate enriched with rare amino acids. The present study clearly shows that the degradation of the feather by YNDH keratinase provided by Laceyella sacchari is not only economical but also a viable method for the better use of the much disregarded feather wastes. Additionally we observed that the concentrations of all different types of amino acids produced through treatment of feather using keratinase developed by laceyella sacchari YNDH was significantly higher than that in feather hydrolysate produced by the native organism directly.
Separation and collection of feather meal and protein lysate
After optimization of enzymatic degradation of waste feather by YNDH protease/keratinase crude enzyme by Response surface methodology (Box-Behnken Design) the feather meal was settled down, centrifuged and dried at 50°C, where, the supernatant containing protein lysate was lyophilized and consolidate the powder as shown as shown Figure 2.
Protease /keratinase purification
The cell free supernatant developed after cultivation was collected, added to Amicon Ultra centrifugal filter (cutoff of 10kDa) to reduce the volume and remove protein impurities. This step is considered a partial purification step where it increased enzyme specific activity from 166.28U/mg to 296.38U/mg and purification fold by 1.78 as shown in (Table 4).
For ion exchange chromatography the working pH should be at least one higher than the PI of the protein. At this pH value the protein will possess a net charge high enough to bind well to ion exchange resin. The concentrated and desalted cell free supernatant was subject to "QFF" strong anion column chromatography which was equilibrated with 20mM Tris- base buffer pH 8.5. The elution was performed with 1M NaCl in the same buffer. The elution profile (Figure 3) indicates that the protease with keratinolytic activity was detected in fractions 6-8. Upon using "QFF" strong anion column, enzyme was purified to 11.31 fold with specific activity and recovery of 1881.3 U/mg and 14.38%, respectively (Table 4). The pooled fractions were loaded and run on 12, 15 and 17% SDS-PAGE to detect the purity. Fig. 4 illustrates the pattern of protein profile before and after purification indicated by reduction in proteins bands to a 3 protein bands at molecular weight of (6 to 10) kDa (Fig. 4).
Characterization of the partially purified protease/keratinase enzyme
Certain characteristics of the partially purified enzyme under study were evaluated using casein and keratin-azure substrates. Some parameters such as optimum temperature and pH, thermal and pH stability, some enzyme inhibitors/ activators, surfactants, detergents, metal ions, and substrate concentration were investigated.
Temperature and pH optimum
The effect of temperature on the partially purified enzyme was studied by measuring the activity at different temperature values from 40 to 90ºC with an interval of 5ºC and 50 to 90ºC with an interval of 10ºC using casein and keratin-azure as substrate, respectively. The results presented in Figure 5 showed that the optimum temperature of the partially purified enzyme when measured as both protease and keratinase activity was 70ºC, so it can be classified as a thermo active protease with keratinolytic activity and the three isolated bands could be worked together as a group of keratinase.
While, the effect of pH on partially purified enzyme activity was investigated by determining of the enzyme activity, first as protease at pH values (7.6–11.6); and second as keratinse at pH values (9.6–10.8). The pH activity profile of the enzyme (protease/keratinase) indicates that it has a broad pH range with an optimum at pH 10.4 as shown in Fig. 6.
Thermal and pH stability
In the present experiment, The effect of temperature on the thermal stability of partially purified enzyme was studied by exposing it to a range of temperatures (50-55ºC) for 24h and (60,70 and75 ºC) for 4h then the residual activity was calculated where (100%) is considered the activity of enzyme kept at room temperature. The enzyme activity was measured as described before and expressed in percent residual activity using casein/keratin-azure substrates (Data not shown).
In summary, the results showed that the enzyme was highly activated (238.46% as protease and 185.4% as keratinase) at temperature 50ºC, moreover it still steady at 55ºC up to 24h. However, the enzyme possessed its stability until reached 90min at 60ºC however, it lost about half of its activity at temperature 70ºC (the optimum T for enzyme activity) after 60min. Moreover it was less stable when exposed to higher temperatures (75ºC).
The pH stability of the partially purified enzyme was investigated as protease and keratinase by measuring the residual activities after incubating the enzyme at pH ranging from 9.6 to 10.8 at room temperature for 60min with time interval of 10min and using casein and keratin azure as substrates. The obtained results indicated that the enzyme was highly stable in all tested pHs (Data not shown).
Effect of some metal ions,detergent, surfactant, solvents, and activators/ inhibitors
The effect of some metal ions on the partial purified protease enzyme activity was examined by measuring the residual activity in the presence of (1mM, 5mM, 10mM, 15mM and 20mM) of each metal ion. All metal ions at concentrations (1, 5 and 10mM) significantly activated protease activity while Hg+2 and Cu+2 inhibited the enzyme to 3.3% and 18.3%, respectively when measured as protease. Moreover, the most significant metals activated the proteolytic activity are Mg+2 and Fe+2 where, Mg+2 significantly increased protease activity to 274.477 and 232.11 at 15mM and 20mM, respectively. While Fe+2 significantly activated protease activity to 411.11% and 1577%, at 15mM and 20mM, respectively as shown in Table 5. The response of studied enzyme as keratinase was investigated towards the previously mentioned; metal ions; detergent, surfactant, solvents; activators/inhibitors, at the most significant concentration (20mM, 2%& 10Mm), respectively. As shown in Table 6; keratinolytic activity of the enzyme significantly activated (410.1% &179.33%) by, Fe+2 and Mg+2, respectively.
On the other hand, the enzyme activity was investigated as protease and keratinase activity in the presence of some surfactants, detergents and solvents. The results represented in Table 5 indicated that a slight increase in the activity of partially purified protease/keratiinase enzyme when treated with Tween-20 and Tween-80 at different concentrations, where Tween-40 highly activated the proteolytic activity and keratiolytic activities to 149.3% and 205.4%, respectively. A sharp increase in proteolytic activity with 219.6% was resulted by incubating the enzyme with TritonX-100 at (1%), but it did not show any effect on keratinolytic activity. However, sharp decrease in the proteolytic/keratinilytic activities was resulted by incubating the enzyme with SDS. Moreover, ethanol, methanol and glycerol had no effect on protease/keratinase enzyme activity while glycerol activated keratinase activity.
Also, the enzyme activity was investigated in the presence of some activators and inhibitors such as EDTA (inhibitor of metallo-type proteases), DTT (reducing agent) and PMSF (a serine protease inhibitor), the results represented in Table 5& 6 showed that both EDTA and DTT caused a slight increase in activity of the partially purified protease/keratinase when measured by both casein and keratin azure. While, PMSF a serine protease inhibitor significantly increased enzyme activity as both protease and keratinase at concentration of 10mM to 120.13% and 176.07%, respectively.
Effect of substrate concentrations
The kinetic parameters (Km and Vmax) of the extracellular YNDH protease/keratinase enzyme for hydrolysis of casein and keratin-azure at 70°C and pH10.4 were determined by double reciprocal Lineweaver-Burk plot. Hydrolysis efficiency represented by Km and Vmax for casein is shown in Fig. 7A and these are 7mg/ml and 384.6 U/mg, respectively. While, the hydrolysis efficiency represented by Km and Vmax for keratin azure is shown in Fig. 7B, these are 7.2mg/ml and 103U/mg, respectively. The estimated Km value indicated the affinity of enzyme towards the substrate, while Vmax is an indication of the catalytic activity of an enzyme which is usually desired to be as high as possible; the high Vmax estimated indicates the high efficiency of tested enzyme towards the substrate.